TABLE 1.

RP-HPLC analysis of the extent of AA oxygenation at high ratios of huPGHS-1 to AA

	Unreacted [1-14C]Arachidonic Acid Remaining After 8 Min(% of Starting Radioactivity)
Reaction Components	Reaction 1	Reaction 2	Reaction 3	Average ± SD
1 μM huPGHS-1 + 1 μM [1-14C]AA no unlabeled AA after 4 min	10	12	9.7	11 ± 1.2
	11	10	12
1 μM huPGHS-1 + 1 μM [1-14C]AA 60 μM unlabeled AA after 4 min	2.4	2.5	2.3	2.4 ± 0.06a
0.1 μM huPGHS-1 + 1μM [1-14C]AA 60 μM unlabeled AA after 4 min	0.88	1.1	1.0	1.0 ± 0.12a
2 μM huPGHS-1 + [1-14C]AA 60 μM unlabeled AA after 4 min	4.8	4.9	4.9	4.9 ± 0.09a

[1-¹⁴C]AA (1 μM) was incubated with the indicated concentration of huPGHS-1 at 37°C for 4 min. Where indicated, 60 μM unlabeled AA was added 4 min after initiating the reaction, and the incubation was continued for another 4 min. The reactions were stopped by the addition of ethyl acetate/acetic acid (20:1), and an aliquot of the organic phase was subjected to radio-reverse-phase (radio-RP-HPLC) to separate the radioactive products and unreacted AA, as described in Experimental Procedures. The results are shown as the percentage of ¹⁴C label remaining in the HPLC fraction eluting with unreacted AA.

^aSignificant difference from control to which no unlabeled AA was added at 4 min in Student t-test (P < 0.005).